DESCRIPTION (Adapted from the investigators abstract): The goal of this project is to increase the understanding of protein structure function relationships, with the long term objective of eventually being able to design drugs that specifically inhibit certain enzymes, as well as redesigning enzymes to perform specific functions. The ability to synthesize better inhibitors would have significant impact on therapeutic approaches to different diseases. The specific aim here is to study the structure-function relationships in the E. coli lac repressor and in both E. coli and human thymidylate synthetase, which has been cloned and expressed in E. coli. The approach to be used takes advantage of recent technological advances that make possible the creation of a very large number of amino acid replacements in a protein by employing synthetic nonsense suppressors operating at nonsense sites. It is now possible to insert 13 different amino acids, in series, at each position in a protein corresponding to a UAG (amber) codon, by using a bank of E. coli nonsense suppressors, many of which have been produced by gene synthesis in vitro. Each amber suppressor inserts one specific amino acid at an amber site. By constructing a series of amber mutations in the gene of choice, using both in vivo and in vitro mutagenesis techniques, a virtual systematic replacement of amino acids can be effected. The effects of the replacements on protein function will be measured and considered in terms of the structural alteration in the protein. These comparisons will be aided by the three-dimensional (3D) structures of the respective proteins. The 3D structure of E. coli thymidylate synthetase in combination with substrate and cofactor inhibitors has been solved, and both the human thymidylate synthetase and the E. coli lac repressor have been crystallized in a form that will lead to the solution of their 3D structures. Additional experiments, including the use of second-site revertants, will be employed to pinpoint important contacts in the protein, as will attempts to change the specificity of ligand binding for each protein under study.